RNA interference (RNAi) employing short double-stranded RNA (siRNA) is a powerful tool for silencing gene expression in mammalian cells (see for example, U.S. Pat. No. 6,506,559, International Publication No. WO 01/29058, International Publication No. WO 01/68836, International Publication No. WO 01/75164, U.S. Publication No. 20020114784, U.S. Publication No. 20030125281, U.S. Publication No. 2002162126, U.S. Publication No. 20030108923, U.S. Publication No. 20020173478, Fire, et al. Nature 391:806-811 (1998); Yang, et al., Mol. Cell. Biol. 21:7807-7816 (2001), Elbashir, et al., Nature 411:494-498 (2001), Hammond et al. Nat. Rev. Genet 2:110-119 (2001), Sharp, Genes Dev. 15:485-490 (2001)).
A standard method for generating siRNA relies on an inherently expensive chemical synthesis of a pre-determined short sequence. Because not all parts of a target sequence are equally effective in silencing, it is necessary to generate libraries of chemically synthesized fragments to identify those sequences which are effective (Holen et al. Nucleic Acids Res. 30:1757-1766, 2002)).
An alternative method for generating siRNA relies on in vitro transcription (see for example, Donze and Picard, Nucleic Acids Res. 30:1757-1766 (2002) and Paddison et al. Genes and Dev. 16:948-958 (2002)). While this approach does not require chemical synthesis it remains necessary to choose and test individual short sequences to determine which are most effective.
Several enzymatic approaches have been reported for cleaving double-stranded RNA molecules into short fragments. An evolutionarily conserved enzyme which is believed to cleave large dsRNA to produce siRNA in vivo has been identified as DICER. (Bernstein, et al., Nature 409:363-366 (2001)). This enzyme contains a helicase motif, a PAZ (PIWI-ARGONAUT-ZWILLE) domain and a tandem repeat of a catalytic domain which is RNaseIII-like. Drosophila extracts presumably containing DICER mixed with large dsRNA in vitro produce short dsRNA in a range of sizes. The preferred size for RNAi applications in this mixture was determined by Tuschl et al. to be 21-23 nucleotides (International Publication No. WO 01/75164). Problems associated with using crude cell extracts containing a putative cleavage enzyme are for example, that it is unclear what proteins in the mixture of proteins are necessary and sufficient to generate the observed effect. In addition, the extract is relatively inefficient at cleaving large double-stranded RNA with only a relatively small amount of the starting material being cleaved to the desired size in vitro even under extended incubation times. (Paddison et al., Proc. Natl. Acad. Sci. 99:1443 (2002)).
More recently, mammalian Dicer has been obtained recombinantly from baculovirus cell expression systems. Lysates of recombinant DICER produced in baculovirus infected insect cell cultures are reported to generate short double-stranded RNA fragments from large double-stranded RNA in the presence of a magnesium buffer. The purified siRNA fragments were used for “silencing” the expression of cognate genes in cultured mammalian cell lines (Myers et al. Nature Biotechnology, 21:324-328 (2003)). Limitations of this approach include the cost of baculovirus expression systems, the incomplete digestion of double-stranded RNA starting material and the need for gel based or other purification step to eliminate precursor RNA prior to performing silencing experiments.
An alternative enzymatic approach for generating small double-stranded RNAs has been to use E. coli RNaseIII in the presence of magnesium ions to partially digest large double-stranded RNA. (Yang et al. Proc. Nat'l. Acad. Sci. USA 99:9942-9947 (2002)). Problems associated with this approach include low recovery amounts of the double-stranded fragments in a specific size range larger than about 15 nt and the associated inconvenience of titration to avoid over or under-digestion. Unless digestion is carefully monitored, RNaseIII in the presence of magnesium ions cleaves large double-stranded RNA into very small fragments that are generally considered to have no known use in RNAi. Careful titration and timing of the partial digest at best yielded a smear throughout a gel after which, a particular size fraction could be recovered for use in RNA silencing in cultured mammalian cells (Yang, et al., Proc. Nat'l. Acad. Sci. USA 99:9942-9947 (2002)). A problem with this approach is the lack of certainty with respect to (a) an end product where the end product relates to yield of a dsRNA having a particular size larger than about 15 nucleotides and (b) the extent of representation of the large double-strand RNA sequence in the cleavage products. The latter may be important since not all parts of the sequence of a long double-stranded RNA are thought to be equally effective in gene silencing and important sequences may be under-represented while unimportant sequences may be over-represented.
Because gene silencing has become a methodology of great importance in understanding molecular functions in cells and organisms, it is desirable to have a rapid, cost effective and reliable method for generating double-stranded RNA suitable for silencing of any gene.